Project Summary/Abstract It is intriguing how a single fertilized egg divides and gives rise to an organism containing a diverse array of cells, tissues, and organs with beautiful three-dimensional (3D) architecture in a precise manner. The forebrain is of particular interest because it is highly-specialized structure with features that are markedly different between species. For example, the neocortex in primates is enormously increased in size and complexity, which probably endow humans with remarkable sensory activities and intellectual ability such as abstract thinking and creativity. Understanding human corticogenesis is important to discover the underlying causes of human-specific diseases such as neurodevelopmental, neurodegenerative, and neuropsychiatric disorders. We ideally need a human brain model. However, due to limited access to human fetal brain tissue and ethical concerns, it has been challenging to directly study human development. Consequently, considerable attention has been placed on the generation of in vitro models using human pluripotent stem cells (hPSCs) to recapitulate aspects of human development and disease. The cerebral organoid is a 3D cortical tissue derived from hPSCs and recapitulates laminar organization of the developing cerebral neocortex in vivo. The advent of such organoid techniques has opened the door for studies of human specific developmental features and paves the way for disease modeling without the use of human fetal tissues. However, many organoid differentiation protocols are inefficient and inconsistent and display marked variability in their ability to recapitulate the 3D architecture and course of neurogenesis in the developing human brain. The goal of my parent K99 is to understand 1) the state of hPSCs that can predict efficient and successful organoid differentiation, 2) to use this robust organoid system to uncover microcircuit formation that has the underlying importance for human brain activities and its malfunction is likely link to neuropsychiatric disorders, such as autism, and 3) to study Fragile X Syndrome, the most common heritable form of cognitive impairment, using human cortical organoids that can give some human-specific insights into mechanisms and cures of this disease. In this supplement application, I will introduce cortical-ganglionic eminence fused-organoids to study the full inhibitory and excitatory neuronal microcircuits to particularly focus on the third aim to model Fragile X Syndrome, using patient derived FXS iPSCs.